PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland

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1 AD Award Number: W81XWH TITLE: Epstein Barr virus and blood brain barrier in Multiple Sclerosis PRINCIPAL INVESTIGATOR: Luwen Zhang CONTRACTING ORGANIZATION: University of Nebraska Lincoln, NE REPORT DATE: January 2014 TYPE OF REPORT: Final report PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.

2 REPORT DOCUMENTATION PAGE Form Approved OMB No Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports ( ), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED Final January TITLE AND SUBTITLE Epstein Barr virus and blood brain barrier in Multiple Sclerosis 01 July December a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Luwen Zhang 5e. TASK NUMBER 5f. WORK UNIT NUMBER lzhang2@unl.edu 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT University of Nebraska 238 Morrison 4240 Fair St Lincoln, NE SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland SPONSOR/MONITOR S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Multiple sclerosis (MS) is a chronic, autoimmune neurodegenerative disease. Epstein-Barr virus (EBV) infection is associated with MS pathogenesis. However, mechanism for the EBV-MS connection is unclear. The blood brain barrier (BBB) is a separation of circulating blood and the brain extracellular fluid in the central nervous system. BBB contains both endothelial cells as well as astrocytes. Interestingly EBV is able to infect both kinds of cells. Because EBV is able to transfer infection from one cell type to another cell type, it is thus hypothesized that EBV uses endothelial cells to infect astrocytes in the BBB, generate serials of cytokines that may eventually cause a leakage in the BBB. An in vitro model for human BBB will be established and infected the endothelial side of the BBB with EBV. Whether BBB is infected by the virus, the expression profiles of cytokines and other cellular genes, as well as the integrity of the BBB will be determined. Because both cytokine production and leakage of BBB are critical events for MS, this line of experiments may provide an evidence to support the etiological role of EBV in MS disease. 15. SUBJECT TERMS Blood-brain-barrier, Epstein-Barr virus; EBV; BBB; MS, Multiple sclerosis 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT a. REPORT U b. ABSTRACT U 18. NUMBER OF PAGES c. THIS PAGE U UU 6 19a. NAME OF RESPONSIBLE PERSON 19b. USAMRMC TELEPHONE NUMBER (include area code)

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4 Introduction: Multiple sclerosis (MS) is a chronic, autoimmune neurodegenerative disease. Many aspects of MS, including its cause, are not well understood. Patients with MS seem to have genetic vulnerability to certain environmental factors such as Epstein-Barr virus (EBV) infections. EBV is a herpesvirus that infects many cell types and associated with other autoimmune diseases. The blood-brain barrier (BBB) is a separation of circulating blood and the brain extracellular fluid in the central nervous system (CNS). The BBB barrier includes endothelial cells, a thick basement membrane, and astrocytes. The BBB leakage is a critical event in MS. Astrocytes maybe one of the major cytokine producers in MS. We hypothesize that EBV uses endothelial cells to infect/bind to astrocytes in BBB, generate serials of cytokines including tumor necrosis factor alpha (TNF-a) and interleukin6 (IL-6) in astrocytes. The production of cytokines may eventually cause a leakage in BBB and allowing other cells/materials into the CNS, leading to nerve cell damages, and eventually MS. Body: All research was done at the University of Nebraska-Lincoln. The objective of this project is to determine if EBV infects astrocytes in the human blood brain barrier (BBB), leading to cytokine production, BBB damage, and eventually multiple sclerosis. Task1: Determine the extent to which EBV infects astrocytes in BBB, leading to cytokine production. EBV may use endothelial cells to infect/bind to astrocytes in human BBB, generating serials of cytokines including tumor necrosis factor alpha (TNF- ) and interleukin 6 (IL-6) in astrocytes. Subtask a: Establish a human BBB model in vitro. We were trying to use primary human brain microvascular endothelial cells and primary astrocytes, and establish a BBB that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other as shown in the following Figure 1. Figure 1: the experimental procedure for establishing a human BBB in vitro. 4

5 We used Matrigel and a mixture of collagen types I and IV as the basement membrane substitutes for our in vitro BBB models. For Matrigel coating, Matrigel was diluted in Dulbecco s PBS (DPBS) to a concentration of 476 μg/ml. The Transwell filter was reversed and 200 μl of diluted Matrigel solution was added to the abluminal side of each Transwell filter. The Transwell plate containing the filters was placed in a 37 C, 5% CO 2-95% air incubator for 3 h for the Matrigel to gel. For collagen mixture coating, types I and IV were diluted in DPBS to 0.5 mg/ml and their ph brought to 7.4 with NaOH and HCl respectively. The two collagens were then mixed thoroughly (40:60, collagen I: collagen IV, v/v) and 200 μl of the mixture was added to the luminal side of each Transwell filter. The Transwell plates with filters were incubated at 37 C for 1 h for the mixture to gel. After that, the Matrigel and collagen mixture coated Transwell filters were placed into a sterile hood and allowed to air dry for tw days. Once dry, the coated filters were rehydrated in DMEM+10% FBS for 1 h in a 37 C, 5%CO 2 95% air incubator prior to cell seeding. Transwell filter with 0.4 μm diameter pores was used as a scaffold for the co-culturing primary human brain endothelial cells and primary human astrocytes on different sides of the filter. The brain microvascular endothelial cells (MEC) were first seeded onto the abluminal side of the inverted Transwell filter at a density of cells per filter (b), allowed to adhere for 2 h (c), then the filter was flipped back and the astrocytes were cultured for 2 days in Astrocyte Medium supplemented with 10% FBS and 1% PS (d). At the end of the second day, Human Brain Microvascular Endothelial Cells (HBMEC) were seeded onto the luminal side of Transwell filter at a density of cells per filter (e) and cocultured with astrocytes for an additional 3 4 days in Endothelial Cell Medium or Astrocyte medium (f). After several days, we then examined the cell attachments to both sides: astrocytes always had big problems! Cells could only cover the surface sparsely: it was independent of the amount of cells introduced. Endothelial cells are better. We were following the published report on the BBB in vitro modeling (Li et al., 2010). Unfortunately, we were unable to establish an unleaky BBB in the laboratory. We have the following explanation for the failure: 1) Human systems are different from rodent system. All established BBB models are using rodent system; 2) Human astrocytes may be too old. The human astrocytes were obtained from adult brain tissue, according to the suppliers. In rodent system, the astrocytes were obtained from 8-10 days old mice (Li et al., 2010). 3) We also did to put the endothelial cells first, and then human astrocytes. Both gave similar results. Subtask 1b: Infect endothelial side of BBB with free virus. Because we failed to establish a functional human BBB model in vitro, we did test if the EBV infects the human brain microvascular endothelial cells. The results showed that EBV can barely infect the cell type. We could not detect any infection by the EBV by Western blot, or GFP expression (Our EBV has a green fluorescent protein inside the genome). However, it has to be stressed that infection of one cell type may not represent the in vivo situation of the BBB. Task2: Determine the extent to which EBV damages BBB. As the human BBB model cannot be established, currently we have not addressed this task yet. We used EBV to infect tightly grown cells; those cells were nonproliferating, as well as sparsely grown cells which are proliferating. EBV may infect endothelial and astrocytes under this proliferating condition. Bothe cells were grown together to mimic the BBB situation in vivo. KEY RESEARCH ACCOMPLISHMENTS: None. Human blood-brain barrier (BBB) in vitro model was not established and we are sorry. However, we did the experiments proposed in the proposal, and we found that EBV may infect the proliferating endothelial and astrocytes, however in the case of non-proliferating, EBV hardly infects any cells! From the conclusion, we infer that EBV is unlikely to infect BBB, as in the BBB, both endothelials and astrocytes are not proliferating. REPORTABLE OUTCOMES: No Publication/Presentation directly resulting from the award. 5

6 CONCLUSION: One major experiment proposed in the grant: to establish and infect the human BBB model and test the gene expression as well as BBB integrity in vitro. This study may establish a role of EBV on MS or other CNS diseases via modulation of BBB as both cytokine production and leakage of BBB are critical events for MS. The proposal is targeting virus infection on both events. We have done the experiments: human BBB model has serious problems. The integrity of the BBB could not be established in our laboratory. This may mean the human cells are different from rodent cells, as rodent BBB has been reported, but not humans. However, we did the infection of co-cultured endothelials and astrocytes in the ability to be infected by EBV. As the quality of BBB has some issues, we did the experiments on alternative approaches. We found that the infection of human brain endothelial cells by EBV seems very difficult. It may mean that EBV may use other routes to gain the entrance of the brain. We used EBV to infect astrocytes in the absence and presence of endothelial cells and test if the endothelial cells affects the capability of EBV to infect astrocytes and furthermore the expression of host genes, especially cytokine genes in astrocytes. The results indicated that both cells are hardly infectable by EBV. In addition, when both cells were in proliferating stage (the plates were not fully covered), EBV may infect at a very low rate as determined by GFP expression. However in the case of nonproliferating conditions, EBV hardly infects any cells! We infer that EBV is unlikely to infect BBB, as in the BBB, both endothelials and astrocytes are not proliferating. REFERENCES: List all references pertinent to the report using a standard journal format (i.e. format used in Science, Military Medicine, etc.). Li, G., Simon, M. J., Cancel, L. M., Shi, Z. D., Ji, X., Tarbell, J. M., Morrison, B., 3rd, and Fu, B. M. (2010). Permeability of endothelial and astrocyte cocultures: in vitro blood brain barrier models for drug delivery studies. Ann Biomed Eng 38(8), APPENDICES: Non-applicable. SUPPORTING DATA: Non-applicable. 6